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T.O. Muldoon, R.B. Rosen, P. Tone, D.–N. Hu; Melatonin Suppression of TPA–Induced VEGF Up–Regulation in Cultured Human Uveal Melanocytes . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5624.
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Up–regulation of VEGF production by 12–0–tetradecanoyl phorbol 13–acetate (TPA) has been demonstrated in a variety cells lines,but has not been previously reported in choroidal melanocytes. Melatonin membrane receptors (Mel 1b) have been detected in human choroidal melanocytes (J. Pineal Res 2000; 28:165), but the effects of melatonin on the expression of VEGF by uveal melanocytes is unknown. The purpose of this study was to investigate the effects of TPA and melatonin on VEGF production by cultured choroidal melanocytes.
Human choroidal melanocytes were isolated from donor eyes and cultured with H16 medium as described previously (Invest Ophthalmol Vis Sci 1993; 33:2210). Early passages of melanocytes were seeded into 12–well plates and cultured with H16 medium (with 2% serum) with or without TPA (100 ng/ml) and melatonin (10–7 M). After 48 hours, conditioned medium was collected and the amount of VEGF was measured using a sandwich enzyme–linked immunosorbent assay kit (R & D Systems).
VEGF could be detected in the conditioned medium from choroidal melanocytes (260 pg/ml). TPA stimulated the production of VEGF (481 pg/ml, p 0.05). However, melatonin significantly reduced the VEGF level in conditioned medium from cells cultured with TPA (296 pg/ml), the difference between cells cultured with TPA alone and TPA with melatonin was statistically significant (p < 0.01).
Several agents, e.g. retinoic acid can down–regulate the TPA–mediated VEGF production by various cells (J Invest Dermatol 1998; 111:907), although they does not decrease the VEGF production in cells not treated with TPA. TPA significantly stimulates the production of VEGF by cultured choroidal melanocytes. Melatonin does not inhibit normal production of VEGF by melanocytes but can suppress the TPA induced up–regulation of VEGF by human uveal melanocytes in vitro.
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